African animal trypanosomosis (AAT) is one of the major diseases of livestock in Côte d'Ivoire. However, the lack of comprehensive, nationally aggregated data hinders the rational planning of control measures and the estimation of their impacts on disease transmission. To address this shortcoming, the Institut Pierre Richet (IPR, Ministry of Health) and the Directorate of Veterinary Services (Ministry of Livestock) embarked on the development of a national tsetse flies and AAT in Côte d'Ivoire. The atlas is a dynamic, georeferenced database on the distribution of disease and its vectors, and this article focuses on the tsetse component of the initiative.All data on tsetse flies collected by the IPR between 2005 and 2024 were retrospectively compiled, georeferenced and harmonised. The data originate from 20 of the 31 regions of the country, and from 3164 sites, corresponding to 5696 trapping events and an overall trapping intensity of 13,181 trap days. Monoconical and, to a lesser extent, biconical traps were used. A total of 27,493 tsetse flies belonging to eight species from the three groups of Glossina were captured. In addition to being the most abundant species, G. palpalis (84.36% of the captures), was also the one with the widest distribution across the country (i.e. 19 of the 20 study regions). The atlas produced provides important evidence for planning tsetse surveillance and monitoring control activities at the national level. However, as next steps, it will be crucial to include also data collected by other stakeholders, to extend surveys to areas of the country not yet covered, and to enhance, finalize and publish the already developed AAT component of the atlas.

The Republic of Guinea has faced an important challenge with human African trypanosomiasis (HAT), which was endemic over the last century. After initial control in the 1960s-1970s, HAT resurged in the 1990s along the Guinean coast, driven by economic and demographic pressures on the mangrove ecosystem. In response, the Guinean government established a national control program in 2002, focusing on medical mass screenings. In 2012, vector control using tiny targets was introduced in the East Boffa focus to reduce fly density and human-vector contact. However, the Ebola epidemic from 2013 to 2016 disrupted these efforts, leading to a reliance on passive screening. Resuming screenings in 2016-2017 revealed increased cases in all foci except the East Boffa area, where vector control had been effective. Vector control continued during the SARS-CoV2 pandemic and at the same time targeted door-to-door screenings were introduced to target high-risk individuals. Since 2018, around 30,000 at-risk individuals have been screened annually. These strategies reduced the total number of new cases below 1 per 10,000 inhabitants in endemic areas over the period 2019-2023, allowing to validate the elimination of HAT as a public health problem. The Guinean team and partners then focused on systematic spatial monitoring of patients and community engagement in vector control. The program also integrates control of other neglected tropical diseases and addresses new research questions, especially about anatomical and animal reservoirs for parasites. These efforts, combined with implementation of improved diagnostic tests and new oral treatments, through active involvement in multiple clinical trials and studies, now aim to interrupt HAT transmission by 2030.

Human leukocyte antigen-G (HLA-G) is an immunomodulatory molecule known to play a crucial role in immune tolerance and regulation. In the context of human African trypanosomiasis (HAT), higher soluble HLA-G levels were detected in the plasma of confirmed cases, representing a serological marker of T. b. gambiense infection. As trypanosomes also invade extravascular tissues, especially the skin, this study explored the potential role of HLA-G in the dermal immune response during T. b. gambiense infection. Blood and skin samples from 50 seronegative individuals, 45 seropositive suspects and 36 confirmed HAT cases, collected between 2018 and 2022 in endemic foci of Guinea and Côte d'Ivoire, were analyzed. Plasmatic and dermal levels of HLA-G proteins were quantified by ELISA and immuno-histochemistry, respectively, and compared to the trypanosome detection results in the same samples. The implication of soluble HLA-G plasma level as a biomarker of T. b. gambiense infection was confirmed. In the dermis, HLA-G isoforms were expressed either with a granular distribution or with in diffuse halos. Granular patterns of dermal HLA-G were directly associated with the presence of trypanosomes in the dermis. The presence of diffuse halos was correlated to higher sHLA-G levels in the plasma. In total, this study provides the first evidence of the involvement of HLA-G in the extravascular immune response against parasites, especially in the skin. It shows that HLA-G distribution in the extravascular compartment also represents a biomarker of trypanosome infection.

In the population at risk of gambiense human African trypanosomiasis (gHAT), the prevalence of extravascular parasite carriage remains unclear. Here, we conducted an observational clinical study in the hypo-endemic gHAT foci of Sinfra and Bonon in Côte d'Ivoire from 2019 to 2022. A total of 74 individuals were enrolled, including 45 suspects previously found positive at least once in a serological test for gHAT and followed by the national elimination programme of Côte d'Ivoire, as well as 29 seronegative controls. No significant differences between groups were observed for any epidemiological parameters and any clinical parameters at enrolment. Whereas trypanosome DNA was detected in the blood of 0/29 controls and 2/45 suspects, the presence of extravascular dermal trypanosomes was confirmed by immuno-histochemistry (fixed trypanosome cells) and/or PCR (trypanosome DNA) in about 1/3 of the suspects (14/45, 31%). However, no gambiense-specific test was found positive in the present study. Hence, the skin could represent an anatomical reservoir for African trypanosomes sustaining a low level of transmission in hypo-endemic foci.

Elimination of gambiense human African trypanosomiasis (gHAT) as a public health problem has been reached or is in sight in a number of endemic foci and the next step is now to reach the elimination of transmission. The ability to detect Trypanosoma brucei gambiense (T. b. gambiense) in both the last human cases and in a suspected animal reservoir becomes increasingly important to reach this goal. We have evaluated here the diagnostic performance of the AnTat A/B and LiTat A/B primers in comparison with the TBR, TgsGP and nested TgsGP PCRs that are currently used for the molecular diagnosis of gHAT. The evaluation was based on serial DNA dilutions from two T. b. gambiense strains for sensitivity, purified reference strains for specificity and field strains isolated from pigs in Cote d'Ivoire for field application. Results showed that the two PCRs (AnTat A/B and LiTat A/B) are not specific for T. b. gambiense, limiting their relevance for studies on suspected animal reservoirs. However, they could represent complementary tools to improve the molecular diagnosis of gHAT in the elimination process even if the detection limit was lowest than for the TgsGP PCR. The results also once more suggest that nested TgsGP PCR should be interpreted with caution as they may lead to an over-estimation of the T. b. gambiense prevalence particularly in animal studies.

Vector control (VC) is one of the strategies employed to manage African trypanosomoses. This study aimed at assessing the effectiveness of a VC campaign against Glossina palpalis palpalis using tiny targets (TTs) impregnated with insecticide in an isolated, protected forest in Abidjan, Côte d'Ivoire, while considering ecological, genetic, and operational factors. Between January 2020 and September 2022, 2,712 TTs were deployed at 684 sites, covering a total area of 1.7 km2. VC monitoring was conducted using Vavoua traps during 12 evaluation surveys, between June 2020 and March 2023. Five months after the initial TT deployment, tsetse fly density had decreased by 98.53%. Although tsetse density remained low due to TT redeployment and reinforcement, there was a significant increase a few months after the last redeployment. VC appeared to have minimal impact on the genetic structuring of G. p. palpalis. This suggested recruitment of local surviving tsetse flies all along the VC campaign due to a low probability of tsetse coming into contact with TTs, or to the evolution of behavioral or physiological resistance to control efforts. The genetic study revealed that one of the microsatellite markers used, the GPCAG locus, exhibited a selection signature possibly in response to VC. This could partly explain the challenges encountered in eliminating a seemingly isolated tsetse population thriving in a particularly favorable habitat.

Animal African trypanosomosis (AAT), caused by protist parasites of the genus Trypanosoma, puts upward of a million head of livestock at risk across 37 countries in Africa. The economic impact of AAT and the presence of human-infectious trypanosomes in animals place a clear importance on improving diagnostics for animal trypanosomes to map the distribution of the veterinary parasites and identify reservoirs of human-infectious trypanosomes. We have adapted the CRISPR-based detection toolkit SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) for trypanosomatid parasites responsible for AAT (SHERLOCK4AAT) including Pan-trypanosomatid, Trypanozoon, T. vivax, T. congolense, T. theileri, T. simiae and T. suis assays. To test the applicability of this technique in the field, we analysed dried blood spots collected from 200 farm and 224 free-ranging pigs in endemic and historical human African trypanosomiasis foci in Guinea and Cote d Ivoire, respectively. The results revealed that SHERLOCK4AAT can detect and discriminate between trypanosome species involved in multiple infections with a high sensitivity. 62.7 % [58.1, 67.3] of pigs were found infected with at least one trypanosome species. T. brucei gambiense, a human-infectious trypanosome, was found in one animal at both sites, highlighting the risk that these animals may act as persistent reservoirs. These data suggest that, due to their proximity to humans and their attractiveness to tsetse flies, pigs could act as sentinels to monitor T. b. gambiense circulation using the SHERLOCK4AAT toolbox. ### Competing Interest Statement The authors have declared no competing interest.

Elimination of gambiense human African trypanosomiasis (gHAT) as a public health problem has been reached or is in sight in a number of endemic foci and the next step is now to reach the elimination of transmission. The ability to detect Trypanosomabruceigambiense (T.b.gambiense) in both the last human cases and in a suspected animal reservoir becomes increasingly important to reach this goal. We have evaluated here the diagnostic performance of the AnTat A/B and LiTat A/B primers in comparison with the TBR, TgsGP and nested TgsGP PCRs that are currently used for the molecular diagnosis of gHAT. The evaluation was based on serial DNA dilutions from two T.b.gambiense strains for sensitivity, purified reference strains for specificity and field strains isolated from pigs in Côte d'Ivoire for field application. Results showed that the two PCRs (AnTat A/B and LiTat A/B) are not specific for T.b.gambiense, limiting their relevance for studies on suspected animal reservoirs. However, they could represent complementary tools to improve the molecular diagnosis of gHAT in the elimination process even if the detection limit was lowest than for the TgsGP PCR. The results also once more suggest that nested TgsGP PCR should be interpreted with caution as they may lead to an over-estimation of the T.b.gambiense prevalence particularly in animal studies.

Human African trypanosomiasis (HAT), or sleeping sickness, is currently targeted for elimination. The etiologic agent of HAT is a trypanosome belonging to the species Trypanosoma brucei ( Tb ) s.l., a unicellular parasite transmitted by tsetse flies. Tb s.l. consists of three subspecies: T. b. brucei ( Tbb ), T. b. gambiense ( Tbg ) and T. b. rhodesiense ( Tbr ). These subspecies are morphologically indistinguishable and classified according to host in which they are found, type of disease and geographical distribution. During the last few decades, there has been considerable effort to genetically characterize Tb s.l. isolated from domestic and wild animals in order to better evaluate the impact of animal reservoirs on the epidemiology of HAT. To assess genetic diversity of Tb s.l. strains circulating in three endemic or historical HAT foci in Côte d’Ivoire, we conducted a population genetics study of these parasites. Biological and isolated stock samples collected from pigs and reference stocks were tested with the primers of the Trypanosoma gambiense- specific-glycoprotein gene (TgsGP) and were genetically characterized with eighteen microsatellite primers. TgsGP positive samples did not fit into Tbg as regard to their microsatellite profile. We also found that in Ivoirian foci, Tbb populations (animal trypanosomes) were structured as several strongly isolated units that propagate clonally. This is in variance with other published data on that subspecies. This study confirms the need to develop better tools to explore the relationships between Tbb and Tbg and to study the epidemiological role of potential animal reservoir for Tbg . ### Competing Interest Statement The authors have declared no competing interest.

The sleeping sickness focus of Bonon was the last one still active at a low endemic level in Côte d’Ivoire. An entomological survey carried out in June 2015 during the rainy season using “Vavoua” traps guided subsequent control activities. Indeed, it improved knowledge of tsetse fly ecology. All the tsetse flies caught (i.e. 1909) belonged to the subspecies Glossina palpalis palpalis (Robineau-Desvoidy, 1830), the major vector of Human African Trypanosomiasis (HAT) in Côte d’Ivoire. In this paper, we looked at the relationship between the apparent density (AD, flies/trap/day) and biotopes. The AD significantly varied according to biotopes, with high density around villages. The trypanosomes overall infection rate (mature and immature) according to microscopic observation was 23.2%. When considering mature infections, the infection rate was 5.5 %. Polymerase chain reaction (PCR) analyses confirmed the presence of Trypanosoma brucei s.l. and Trypanosoma congolense “forest type”. Blood meals analysis using cytochrome b gene sequences revealed that tsetse flies fed on pigs. The edges of the villages seem to constitute preferred habitats for tsetse flies where they are protected from insecticide pressure in the fields, and where they can easily take bloodmeals from free-ranging pigs. The findings of this study provided a baseline in decision-making for subsequent vector control activities.

Animal African trypanosomosis (AAT), caused by protist parasites of the genus Trypanosoma, puts upward of a million head of livestock at risk across 37 countries in Africa. The economic impact of AAT and the presence of human-infectious trypanosomes in animals place a clear importance on improving diagnostics for animal trypanosomes to map the distribution of the veterinary parasites and identify reservoirs of human-infectious trypanosomes. We have adapted the CRISPR-based detection toolkit SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) for trypanosomatid parasites responsible for AAT (SHERLOCK4AAT) including Pan-trypanosomatid, Trypanozoon, T. vivax, T. congolense, T. theileri, T. simiae, and T. suis assays. To test the applicability of this technique in the field, we analysed dried blood spots collected from 200 farm and 224 free-ranging pigs in endemic and historical human African trypanosomiasis foci in Guinea and Côte d’Ivoire, respectively. The results revealed that SHERLOCK4AAT can detect and discriminate between trypanosome species involved in multiple infections with a high sensitivity. 62.7% [58.1, 67.3] of pigs were found infected with at least one trypanosome species. T. brucei gambiense, a human-infectious trypanosome, was found in one animal at both sites, highlighting the risk that these animals may act as persistent reservoirs. These data suggest that, due to their proximity to humans and their attractiveness to tsetse flies, pigs could act as sentinels to monitor T. b. gambiense circulation using the SHERLOCK4AAT toolbox.

Background:Serological screening tests play a crucial role to diagnose gambiense human African trypanosomiasis (gHAT). Presently, they preselect individuals for microscopic confirmation, but in future “screen and treat” strategies they will identify individuals for treatment. Variability in reported specificities, the development of new rapid diagnostic tests (RDT) and the hypothesis that malaria infection may decrease RDT specificity led us to evaluate the specificity of 5 gHAT screening tests. Method: During active screening, venous blood samples from 1095 individuals from Côte d’Ivoire and Guinea were tested consecutively with commercial (Bioline HAT 2.0, HAT Sero-K-SeT, CATT) and prototype (HAT Sero-K-SeT 2.0, DCN) gHAT screening tests and with a malaria RDT. Individuals with ≥ 1 positive gHAT screening test underwent microscopy and further immunological (trypanolysis, indirect ELISA) and molecular laboratory tests (conventional PCR, SHERLOCK, Trypanozoon S²-RT-qPCR, SNP RT-qPCR). Microscopic trypanosome detection confirmed gHAT, while other individuals were considered gHAT free. Results: One gHAT case was diagnosed. Overall test specificities (n=1094) were: CATT 98.9% (98.1-99.4%); HAT Sero-K-SeT 86.7% (84.5-88.5%); Bioline HAT 2.0 82.1% (79.7-84.2%); DCN HAT RDT 78.2% (75.7-80.6%); and HAT Sero-K-SeT 2.0 78.4% (75.9-80.8%). In malaria positives, gHAT screening tests appeared less specific, but the difference was significant only in Guinea for Bioline HAT 2.0 and HAT Sero-K-Set 2.0. The specificities of immunological and molecular laboratory tests in gHAT seropositives were 98.7-100% (n=399) and 93.0-100% (n=302), respectively. Among 44 laboratory test positives, only the confirmed gHAT patient and one screening test seropositive combined immunological and molecular laboratory test positivity. Conclusions:Although a minor effect of malaria cannot be excluded, gHAT RDT specificities are far below the 95% minimal specificity stipulated by the WHO target product profile for a simple diagnostic tool to identify individuals eligible for treatment. Unless specificity is improved, an RDT-based “screen and treat” strategy would result in massive overtreatment. In view of their inconsistent results, additional comparative evaluations of the diagnostic performance of laboratory tests are indicated for better identifying, among screening test positives, those at increased suspicion for gHAT. Trial registration: The trial was retrospectively registered under NCT05466630 in clinicaltrials.gov on July 15 2022.

Trypanosoma brucei gambiense (Tbg) group 2 is a subgroup of trypanosomes able to infect humans and is found in West and Central Africa. Unlike other agents causing sleeping sickness, such as Tbg group 1 and Trypanosoma brucei rhodesiense, Tbg2 lacks the typical molecular markers associated with resistance to human serum. Only 36 strains of Tbg2 have been documented, and therefore, very limited research has been conducted despite their zoonotic nature. Some of these strains are only available in their procyclic form, which hinders human serum resistance assays and mechanistic studies. Furthermore, the understanding of Tbg2’s potential to infect tsetse flies and mammalian hosts is limited. In this study, 165 Glossina palpalis gambiensis flies were experimentally infected with procyclic Tbg2 parasites. It was found that 35 days post-infection, 43 flies out of the 80 still alive were found to be Tbg2 PCR-positive in the saliva. These flies were able to infect 3 out of the 4 mice used for blood-feeding. Dissection revealed that only six flies in fact carried mature infections in their midguts and salivary glands. Importantly, a single fly with a mature infection was sufficient to infect a mammalian host. This Tbg2 transmission success confirms that Tbg2 strains can establish in tsetse flies and infect mammalian hosts. This study describes an effective in vivo protocol for transforming Tbg2 from procyclic to bloodstream form, reproducing the complete Tbg2 cycle from G. p. gambiensis to mice. These findings provide valuable insights into Tbg2’s host infectivity, and will facilitate further research on mechanisms of human serum resistance.

The skin is an anatomical reservoir for African trypanosomes, yet the prevalence of extravascular parasite carriage in the population at risk of gambiense Human African Trypanosomiasis (gHAT) remains unclear. Here, we conducted a prospective observational cohort study in the HAT foci of Forecariah and Boffa, Republic of Guinea. Of the 18,916 subjects serologically screened for gHAT, 96 were enrolled into our study. At enrolment and follow-up visits, participants underwent a dermatological examination and had blood samples and superficial skin snip biopsies taken for examination by molecular and immuno-histological methods. In seropositive individuals, dermatological symptoms were significantly more frequent as compared to seronegative controls. Trypanosoma brucei DNA was detected in the blood of 67% of confirmed cases (22/33) and 9% of unconfirmed seropositive individuals (3/32). However, parasites were detected in the extravascular dermis of up to 71% of confirmed cases (25/35) and 41% of unconfirmed seropositive individuals (13/32) by PCR and/or immuno-histochemistry. Six to twelve months after treatment, trypanosome detection in the skin dropped to 17% of confirmed cases (5/30), whereas up to 25% of unconfirmed, hence untreated, seropositive individuals (4/16) were still found positive. Dermal trypanosomes were observed in subjects from both transmission foci, however, the occurrence of pruritus and the PCR positivity rates were significantly higher in unconfirmed seropositive individuals in Forecariah. The lower sensitivity of superficial skin snip biopsies appeared critical for detecting trypanosomes in the basal dermis. These results are discussed in the context of the planned elimination of gHAT.

Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense cause human African trypanosomiasis (HAT), a neglected tropical disease that constitutes an important public health issue in sub-Saharan Africa. In the absence of a vaccine, only chemotherapy and vector control has been used to combat the disease. Environmental factors, such as exposure to infected tsetse files, and genetic factors such as variants in the APOL1 gene have been shown to contribute to the risk of developing HAT. However, the known factors only explain a small part of the risk of developing trypanosomiasis. We have undertaken a genome wide association study (GWAS) using 3813 samples from T. b. gambiense and T.b. rhodesiense HAT foci in Guinea, Côte d’Ivoire, Cameroon, DRC, Malawi and Uganda. 2141 samples were genotyped on the H3Africa SNP chip followed by a genotyping a validation cohort of an additional 1,627 samples at candidate loci. After the primary and validation studies we identified a novel locus near SMOC2 with genome-wide significance. We also identified suggestive associations near NXN, NTNG1 and NCKAP5 that have stronger associations with disease susceptibility than the APOL1 loci that has been previously identified by hypothesis driven approaches. These genes offer new entry points for future studies of the underlying genetic mechanisms of HAT.### Competing Interest StatementThe authors have declared no competing interest.### Funding StatementThis work was supported by Human Heredity and Health in Africa (H3Africa) programme under Wellcome Trust grant number 099310/Z/12/Z and H3Africa grant number H3A-18- 004. H3Africa is managed by the Science for Africa Foundation (SFA Foundation) in partnership with Wellcome, NIH and AfSHG. The views expressed herein are those of the author(s) and not necessarily those of the SFA Foundation and her partners.### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:The Minister de la Sante Publique (Democratic Republic of Congo) gave ethical approval for this work No 1/2013; Ministere de la Sante et de la Lutte Contre le SIDA of Cote d'Ivoıre gave ethical approval for this work; Uganda Vector Control Division Research Ethics Committee (Ministry of Health), gave ethical approval for this work ; Uganda National Council for Science and Technology HS 1344 gave ethical approval for this work; Comite Consultatif de Deontologie et d'Ethique [CCDE] de l'Institut de Recherche pour le Developpement: gave ethical approval for this work; 1-22/04/2013; Cameroon (Le Comite National d'Ethique de la Recherche pour la Sante Humain: 2013/364/L/CNERSH/ SP), Comite National D'Ethique et de la Recherche 2014/No 38/ MSLS/CNER-dkn) gave ethical approval for this work Malawi National Health Sciences Research Committee, protocol numbers NHSRC15/4/1399 and Malawi 1213 gave ethical approval for this work I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.YesAll data produced in the present study are available upon reasonable request to the authors

Abstract Background Serological screening tests play a crucial role to diagnose gambiense human African trypanosomiasis (gHAT). Presently, they preselect individuals for microscopic confirmation, but in future “screen and treat” strategies they will identify individuals for treatment. Variability in reported specificities, the development of new rapid diagnostic tests (RDT) and the hypothesis that malaria infection may decrease RDT specificity led us to evaluate the specificity of 5 gHAT screening tests. Methods During active screening, venous blood samples from 1095 individuals from Côte d’Ivoire and Guinea were tested consecutively with commercial (CATT, HAT Sero-K-SeT, Abbott Bioline HAT 2.0) and prototype (DCN HAT RDT, HAT Sero-K-SeT 2.0) gHAT screening tests and with a malaria RDT. Individuals with ≥ 1 positive gHAT screening test underwent microscopy and further immunological (trypanolysis with T.b. gambiense LiTat 1.3, 1.5 and 1.6; indirect ELISA/T.b. gambiense; T.b. gambiense inhibition ELISA with T.b. gambiense LiTat 1.3 and 1.5 VSG) and molecular reference laboratory tests (PCR TBRN3, 18S and TgsGP; SHERLOCK 18S Tids, 7SL Zoon, and TgsGP; Trypanozoon S2-RT-qPCR 18S2, 177T, GPI-PLC and TgsGP in multiplex; RT-qPCR DT8, DT9 and TgsGP in multiplex). Microscopic trypanosome detection confirmed gHAT, while other individuals were considered gHAT free. Differences in fractions between groups were assessed by Chi square and differences in specificity between 2 tests on the same individuals by McNemar. Results One gHAT case was diagnosed. Overall test specificities (n = 1094) were: CATT 98.9% (95% CI: 98.1–99.4%); HAT Sero-K-SeT 86.7% (95% CI: 84.5–88.5%); Bioline HAT 2.0 82.1% (95% CI: 79.7–84.2%); DCN HAT RDT 78.2% (95% CI: 75.7–80.6%); and HAT Sero-K-SeT 2.0 78.4% (95% CI: 75.9–80.8%). In malaria positives, gHAT screening tests appeared less specific, but the difference was significant only in Guinea for Abbott Bioline HAT 2.0 (P = 0.03) and HAT Sero-K-Set 2.0 (P = 0.0006). The specificities of immunological and molecular laboratory tests in gHAT seropositives were 98.7–100% (n = 399) and 93.0–100% (n = 302), respectively. Among 44 reference laboratory test positives, only the confirmed gHAT patient and one screening test seropositive combined immunological and molecular reference laboratory test positivity. Conclusions Although a minor effect of malaria cannot be excluded, gHAT RDT specificities are far below the 95% minimal specificity stipulated by the WHO target product profile for a simple diagnostic tool to identify individuals eligible for treatment. Unless specificity is improved, an RDT-based “screen and treat” strategy would result in massive overtreatment. In view of their inconsistent results, additional comparative evaluations of the diagnostic performance of reference laboratory tests are indicated for better identifying, among screening test positives, those at increased suspicion for gHAT. Trial registration The trial was retrospectively registered under NCT05466630 in clinicaltrials.gov on July 15 2022. Graphical Abstract

Background Human African trypanosomiasis (HAT) is an important disease of sub-Saharan Africa that is approaching elimination in many regions. However, the disease has previously returned from similarly low case numbers in the past, making it important to identify issues that hinder elimination efforts. One important factor is likely to be the recent characterization of individuals with latent HAT infections that are able to tolerate HAT with few symptoms and to control blood parasitaemia to levels that are undetectable by microscopy. Although animal trypanotolerance has been examined in detail, it is unclear how the latent phenotype is maintained in humans.Methods To identify immune components involved in latent HAT, we used targeted RNASeq to examine the expression of 495 immune-related transcripts in blood collected from 287 individuals at active disease foci in Guinea. These samples included latent infections, HAT clinical cases, and uninfected controls. The in vivo effects of IL21 functional blockade was investigated using a murine model of trypanosomiasis.Results Differential expression analysis revealed transcripts involved in T cell activation and B cell development that associated with trypanosome infection, including PD1 , CD70 , and CD80 . In particular, IL21 was found to be elevated in infected individuals, although it was significantly higher in clinical cases relative to latent infections. This pattern was replicated at the protein level when patient sera were examined by ELISA. Reducing IL21 pathway activity in mice infected with Trypanosoma brucei led to increased survivorship and reduced parasitaemia in the model animals.Conclusion Our data show that IL21 is a potential biomarker of Human African Trypanosomiasis and is a cause rather than a consequence of symptoms severity. Further investigation of IL21 will contribute to understanding the factors involved in developing latent HAT, improving control efforts to identify and predict such infections. In the future, the factors identified in this study may also serve as intervention targets to control the symptoms of trypanosomiasis.### Competing Interest StatementThe authors have declared no competing interest.### Funding StatementPC, AC, AML were funded by a Wellcome Senior Fellowship to AML (209511/Z/17/Z). BB was funded by IRD. WJK, HN and HI, were supported through the Human Hereditary and Health in Africa (H3Africa) [H3A/18/004]. The second phase of the Wellcome component of H3Africais being implemented by the African Academy of Sciences (AAS) and the NEPAD Agency's Alliance for Accelerating Excellence in Science in Africa (AESA) in partnership with Wellcome.### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:All investigations on humans were conducted in accordance with the Declaration of Helsinki. Participants were identified through healthcare providers, community engagement and active surveillance campaigns led by the national contral program, Ministry of Health Guinea. Written informed consents for sample collection, analysis and publication of anonymised data was obtained from all participants by trained local healthcare workers. Subjects or their legal guardian gave consent as a signature or a thumbprint after receiving standardized information in French or their local langage as preferred. Ethical approvals for the study was obtained from within the TrypanoGEN Project following H3Africa Consortium guidelines for informed consent and from Comite Consultatif de Deontologie et ethique (CCDE) at the Institut de recherche pour le Developpent (IRD; 10/06/2013). Research procedures were also approved by the University of Glasgow MVLS Ethics Committee for Non-Clinical Research Involving Human Subjects (Reference no. 200120043). All animal experiments were approved by the University of Glasgow Ethical Review Committee and performed in accordance with the UK Home Office guidelines, UK Animals (Scientific Procedures) Act, 1986 and EU directive 2010/63/EU. All experiments were conducted under SAPO regulations and UK Home Office project licence number PC8C3B25C to Dr. Jean Rodgers. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.YesAll data produced in the present study are available upon reasonable request to the authors and the TrypanoGEN network (H3Africa)

Although studies on African Trypanosomiases revealed a variety of trypanosome species in the blood of various animal taxa, animal reservoirs of Trypanosoma brucei gambiense and anatomical niches such as skin have been overlooked in most epidemiological settings. This study aims to update epidemiological data on trypanosome infections in animals from human African trypanosomiasis (HAT) foci of Cameroon. Blood and skin snips were collected from 291 domestic and wild animals. DNA was extracted from blood and skin snips and molecular approaches were used to identify different trypanosomes species. Immunohistochemical analyses were used to confirm trypanosome infections in skin snips. PCR revealed 137 animals (47.1%) with at least one trypanosome species in the blood and/or in the skin. Of these 137 animals, 90 (65.7%) and 32 (23.4%) had trypanosome infections respectively in the blood and skin. Fifteen (10.9%) animals had trypanosome infections in both blood and skin snip. Animals from the Campo HAT focus (55.0%) were significantly (X2 = 17.6; P< 0.0001) more infected than those (29.7%) from Bipindi. Trypanosomes of the subgenus Trypanozoon were present in 27.8% of animals while T. vivax, T. congolense forest type and savannah type were detected in 16.5%, 10.3% and 1.4% of animals respectively. Trypanosoma b. gambiense infections were detected in the blood of 7.6% (22/291) of animals. No T. b. gambiense infection was detected in skin. This study highlights the presence of several trypanosome species in the blood and skin of various wild and domestic animals. Skin appeared as an anatomical reservoir for trypanosomes in animals. Despite methodological limitations, pigs, sheep, goats and wild animals were confirmed as potential reservoirs of T. b. gambiense. These animal reservoirs must be considered for the designing of control strategies that will lead to sustainable elimination of HAT.

Human African trypanosomiasis is a life-threatening parasitic infection endemic to sub-Saharan Africa. Around 95% of cases are due to Trypanosoma brucei gambiense, found in western and central Africa. Clinical signs and symptoms are nonspecific, current diagnostic tests are not sufficiently accurate, and parasitological confirmation of infection requires microscopic examination of body fluids and specialized techniques for concentrating parasites. Moreover, current treatment is not recommended on the basis of suspicion alone because it is not sufficiently safe. The availability of a simple and accurate diagnostic test to identify individuals harbouring parasites would widen treatment and help decrease disease prevalence. A subcommittee of the World Health Organization's Neglected Tropical Diseases Diagnostics Technical Advisory Group has developed a target product profile for a diagnostic tool to identify T. b. gambiense infection. This tool should have a high sensitivity for detecting T. b. gambiense but be simple enough to use in rural Africa. Ideally, the tool could be applied by any minimally trained individual in an unsophisticated peripheral health facility, or a mobile team in a village with little infrastructure. The test should be able to function under hot and humid conditions. Basic training should take under 2 hours and the test should involve fewer than five steps. There should be no need for instrumentation or precision liquid handling. The test should yield a qualitative result in under 20 minutes that can be easily observed, and one test should be sufficient for determining treatment. A unit cost below 1 United States dollar (US$) would enable mass screening.